To understand more thoroughly the biosynthesis of vitamin K-dependent clotting factors the aim of the proposed research is to purify and characterize the vitamin K-dependent gamma glutamyl carboxylase. Stabilization of the enzyme with combinations of phospholipid, imidazole, glycerol, and sulfhydryl reagents is planned during purification, potential steps of whih include: salt fractionation, organic solvent fractionation (e.g., alcohol precipitation), DEAE, QAE, CM, hydroxyapatite and cellulose phosphate exchange chromatography, gel filtration, isoelectric focusing, preparative gel electrophoresis, affinity chromatography, high pressure liquid chromatography, sucrose gradient centrifugation, phase partitioning, low pressure hydrophobic column chromatography. Both solubilized liver microsomes (rat, bovine, etc.) and acetone powders of these microsomes will be evaluated as starting material for this purification. Since it has been shown that gamma-carbon-hydrogen bond cleavage of glutamic acid (measured as vitamin K-dependent tritium release from beta, gamma-tritiated glutamic acid in appropriate carboxylase substrates) can be dissociated from carboxylation, during fractionation both tritium release and carboxylation will be assayed for, so that should separation of proteins concerned with each of these functions occur, it might be detected. The purified carboxylase will be studied to determine the detailed mechanism by which vitamin K drives this reaction. In particular, the following are to be evaluated: the stoichiometry, the relationship of the vitamin K epoxidase activity; is a formal carbanion formed at the gamma-carbon prior to carboxylation?; is there a vitamin K-dependent peroxidase activity?; is there an "active oxygen" involved?; is the reaction mechanism a radical-radical C-carboxylation?; are there subunits which catalyze partial reactions?; how do tetrachloropyridinol and the imidazopyridines inhibit? To facilitate these studies carboxylase substrates containing either specifically gamma-tritiated glutamic acid or beta-fluorinated glutamic acid are to be prepared.